NF-KB及相关microRNA在TNFα诱导的猪脂肪细胞凋亡中的调控机理研究

The nuclear transcription factor NF-κB widely exists in various types of cells and there is a close relationship between activated NF-κB and celluar apoptosis, NF-κB may exhibit pro-apoptotic or anti-apoptotic or two-way regulatory role in different cells.Tumor necrosis factor a (TNFa) could induces apoptosis in adipocytes which is an important mechanism for the regulation in fat depot and redistribution in body. Whereas the binding of TNFa to its membrane receptor is an important approach to activate NF-κB, we speculate that NF-κB plays an essential role in adipocytic apoptosis induced by TNFa, but the specific function of NF-κB in this process is to be elucidated. In addition, some studies found that several MicroRNA have significant effects on the activation and expression of NF-κB. Therefore, this study trying to confirm the precise role of NF- κB in porcine adipocyte apoptosis induced by TNFa and further to explores the molecular mechanism. Moreover, the high throughput sequencing is used for functional screen of MicroRNAs related with apoptosis induced by TNFa/NF-κ B and find out some key candidates. Finially, the regulatory and cross-talk between candidated MicroRNA and NF-κ B will be evaluated. These research results will provide an important theoretical help for the studies about animal fat metabolism and related metabolic diseases.

活化的核转录因子NF-κB与细胞凋亡有密切关系，在不同种类的细胞中会表现出促凋亡或者抗凋亡或者双向调控作用。肿瘤坏死因子（TNFa）诱导脂肪细胞凋亡，是机体调整脂肪库的容量大小和脂肪再分布的重要机制。本研究鉴于TNFa与细胞膜受体蛋白结合是激活NF-κB的重要途径，推测NF-κB在TNFa诱导的脂肪细胞凋亡中发挥重要的作用，但其扮演什么样的角色还不清楚。此外，一些MicroRNA对NF-κB的激活和表达也有重要的影响。本项目选用猪作为试验动物，首先研究确认NF-κB在TNFa诱导的猪脂肪细胞凋亡中的确切作用和分子机制；其次使用高通量基因芯片筛选和确认与NF-kB密切相关的microRNA并进行功能验证；然后深入研究候选microRNA的表达水平与NF-κB信号通路和脂肪细胞凋亡之间的相互调控关系和对话机制。相关研究结果对于人和动物脂肪代谢及相关代谢症的研究提供重要理论帮助。

人和动物体内脂肪的过度积累会造成肥胖症和2型糖尿病等代谢疾病，关于脂肪细胞凋亡的研究有助于相关代谢疾病的诊疗。肿瘤坏死因子α（Tumor necrosis factor，TNFα）能够通过死亡受体通路诱导包括脂肪细胞在内多种类型细胞的凋亡。除此以外，TNFα还能够激活细胞中的核转录因子NF-κB，后者能够靶向调控众多功能基因并在不同类型细胞的凋亡中扮演不同的调控角色。但是NF-κB对脂肪细胞凋亡产生怎样的调控作用还不完全清楚。本课题通过大量细胞试验和分子生物学检测明确了TNFα可以诱导分化的脂肪细胞（猪原代脂肪细胞和3T3-L1脂肪细胞系）适度凋亡并快速激活NF-κB，使用特异性阻断剂PDTC抑制NF-κB激活能够明显增加凋亡细胞数量，促进TNFα诱导的脂肪细胞凋亡，这说明活化的NF-κB负调控了TNFa诱导的脂肪细胞凋亡。使用microRNA芯片检测发现TNFα处理能够显著上调细胞中的miRNA-224-5p(miR-224)的表达而PDTC则显著抑制miR-224的表达，提示miR-224与NF-κB的活化有关。使用mimic转染过表达脂肪细胞内的miR-224能够明显增强TNFα对NF-κB的激活作用，进而减少凋亡细胞数量，降低脂肪细胞对TNFα的敏感性。使用inhibitor转染沉默细胞中的miR-224则起到相反的调控作用。靶基因预测和双荧光素酶报告试验确认TRAF-associated NF-κB activator （TANK）基因是miR-224a的靶基因。TANK是NF-κB通路上游效应因子，能够与TRAF竞争性结合从而负调控NF-κB的活化。使用特异性siRNA沉默细胞内源TANK基因能够显著促进TNFa诱导的脂肪细胞凋亡。这些结果说明miR-224通过靶向TANK基因影响NF-κB的活化进而参与TNFα诱导的脂肪细胞凋亡。之前有研究发现miR-199a能够调控脂肪细胞增殖和分化。在一些癌细胞中，miR-199a与NF-κB存在互作关系。本课题研究确认miR-199a能够负调控NF-κB的活化，过表达miR-199a显著抑制NF-κB的激活进而促进TNFα诱导的脂肪细胞凋亡。这些试验结果有助于我们更好的理解脂肪细胞凋亡的分子机制。